Identification and characterization of a ligand-regulated nuclear export signal in androgen receptor

Androgen receptor (AR) belongs to the steroid receptor superfamily that regulates gene expression in a ligand-dependent fashion. AR is localized to the cytoplasm in the absence of androgen and translocates into the nuclei to activate gene expression in the presence of ligand. Regulation of AR nuclear import and export represents an essential step in androgen action. A nuclear localization signal (NLS) has been identified in the DNA-binding domain and hinge region of AR and other steroid receptors. Studies on nuclear export of AR, however, are limited, and what might be the underlying mechanism regulating the intracellular localization of steroid receptors is unclear. Our studies have identified a leptomycin B-insensitive nuclear export signal (NESAR) in the ligand-binding domain of AR, which is active in the absence of androgen and repressed upon ligand binding. Consistent with its androgen-sensitivity, NESAR contains amino acid residues in the immediate vicinity of the bound ligand. NESAR is necessary for AR nuclear export and is dominant over the NLS in the DNA-binding domain and hinge region in the absence of hormone. Our findings suggest that androgen can regulate NESAR and, subsequently, the NLS of the AR, providing a mechanism by which androgen regulates AR nuclear/cytoplasmic shuttling. Estrogen receptor alpha and mineralocorticoid receptor also contain functional NES, suggesting that this ligand-regulated NES is conserved among steroid receptors.     

Molecular cloning of new human TR2 receptors: a class of steroid receptor with multiple ligand-binding domains

Previously we isolated a new group of cDNA clones from human testis cDNA libraries which might code for new steroid receptors. The cDNA and predicted amino acid sequences of two of these receptors, named TR2-5 and TR2-7 receptors, were determined. We report here the nucleotide and deduced amino acid structures of two other receptors that we named TR2-9 and TR2-11 receptors. The calculated MW of TR2-5 receptor, TR2-7 receptor, TR2-9 receptor and TR2-11 receptor are 52,982, 20,528, 50,849 and 67,223 respectively, which match well with the apparent MW of in vitro translated products. The 26 amino acids involved in the formation of "Zn-fingers" are conserved. The ligand-binding domain of TR2-9 receptor is 16 amino acids shorter and has 3 different amino acids compared with TR2-5 receptor. The TR2-11 receptor has a ligand-binding domain which is longer and quite different compared with the other TR2 receptors. The multiple ligand-binding domains of TR2 receptor could be the products of different genes or may be due to RNA splicing errors. So far, we have failed to find binding activity with any known steroid hormone; this promotes the possibility that an unidentified steroid hormone may be involved.     

Comparison of class A and D G protein-coupled receptors: common features in structure and activation

All G protein-coupled receptors (GPCRs) share a common seven TM helix architecture and the ability to activate heterotrimeric G proteins. Nevertheless, these receptors have widely divergent sequences with no significant homology. We present a detailed structure-function comparison of the very divergent Class A and D receptors to address whether there is a common activation mechanism across the GPCR superfamily. The Class A and D receptors are represented by the vertebrate visual pigment rhodopsin and the yeast alpha-factor pheromone receptor Ste2, respectively. Conserved amino acids within each specific receptor class and amino acids where mutation alters receptor function were located in the structures of rhodopsin and Ste2 to assess whether there are functionally equivalent positions or regions within these receptors. We find several general similarities that are quite striking. First, strongly polar amino acids mediate helix interactions. Their mutation generally leads to loss of function or constitutive activity. Second, small and weakly polar amino acids facilitate tight helix packing. Third, proline is essential at similar positions in transmembrane helices 6 and 7 of both receptors. Mapping the specific location of the conserved amino acids and sites of constitutively active mutations identified conserved microdomains on transmembrane helices H3, H6, and H7, suggesting that there are underlying similarities in the mechanism of the widely divergent Class A and Class D receptors.     

Ketoconazole binds to the human androgen receptor.

Ketoconazole, an imidazole anti-fungal agent, has often produced features of androgen deficiency including decreased libido, gynecomastia, impotence, oligospermia, and decreased testosterone levels, in men being treated for chronic mycotic infections. Based on these potent effects on gonadal function in vivo as well as previous work in vitro demonstrating affinity of ketoconazole for receptor proteins for glucocorticoids and 1,25(OH)2 vitamin D3 and for sex steroid binding globulin (SSBG), the binding of ketoconazole to human androgen receptors (AR) in vitro was also examined. Ketoconazole competition with [3H]methyltrienolone (R1881) for androgen binding sites in dispersed, intact cultured human skin fibroblasts was determined at 22 degrees C. Fifty percent displacement of [3H]R1881 binding to AR was achieved by 6.4 +/- 1.8 (SE) x 10(-5) M ketoconazole. Additional binding studies performed with ketoconazole in the presence of increasing amounts of [3H]R1881 showed that the interaction of ketoconazole with AR was competitive when the data were analyzed by the Scatchard method. It should be noted, however, that the dose of ketoconazole required for 50% occupancy of the androgen receptor is not likely to be achieved in vivo, at least in plasma. Finally, androgen binding studies performed with other imidazoles, such as clotrimazole, miconazole, and fluconozole, revealed that in this class of compounds only ketoconazole appears to interact with the androgen receptor. Ketoconazole appears to be the first example of a non-steroidal compound which binds competitively to both SSBG and multiple steroid hormone receptors, suggesting that the ligand binding sites of these proteins share some features in common.     

雄激素受体的作用机制

主要概述了雄激素受体的作用机制,特别对影响雄激素受体特异性的因素进行探讨.雄激素受体（AR）属于甾体激素受体超家族,能通过配体依赖方式与特异的DNA序列结合,调控基因转录.